Abstract Using a new approach, it has been recently found that the recognition of an amino-terminal residue in an intracellular protein mediates the metabolic stability of the protein and apparently also the potential for regulation of its stability (1). The objective of the research described in this proposal is to explore the implications of this insight (the "N-end rule" pathway (1)). Summary of Specific Aims: 1) Construction of ubiquitin fusions with test proteins other than beta-galactosidase (beta gal), in particular fusions with the mouse dihydrofolate reductase (DHFR). Use of these ubiquitin fusions to address the mechanism of targeting by the N-end rule proteolytic pathway. 2) Determination of whether the N-end rule as defined with beta gal is the same when defined with unrelated test proteins. 3) Determination of whether the N-end rule as defined in the yeast S. cerevisiae is the same when defined in E. coli, frog oocytes and cultured mammalian cells. 4) Mapping positions of ubiquitin moieties within a targeted, mulitply ubiquitinated protein. 5) Isolation, purification and molecular genetic analysis of the ubiquitin-specific processing from yeast. Use of this protease for direct testing of the N-end rule in prokaryotes (E. coli). 6) Use of a novel screening approach to isolate yeast mutants in selective protein turnover. 7) Mechanistic studies on selective protein turnover in vitro using both ubiquitin-beta gal fusions (1) and analogous well-defined substrates. 8) Biochemical and molecular genetic analyses of the regulation and function of posttranslational conjugation of amino acids to amino-termini of proteins. Detailed mechanistic understanding of selective protein turnover, still a distant goal, is expected to prove helpful if not essential for solving many of the currently outstanding biological problems, from regulation of cell cycle progression to differentiation, stress responses, carcinogenesis and aging.